Philip A. Schwartzkroin

Calorie restriction and ketogenic diet diminish neuronal excitability in rat dentate gyrus in vivo

Summary:  Purpose: The ketogenic diet (KD) is an effective treatment for intractable epilepsy. However, little is known about its underlying mechanisms.

Physiological and Morphological Characterization of Dentate Granule Cells in the p35 Knock-Out Mouse Hippocampus: Evidence for an Epileptic Circuit

There is a high correlation between pediatric epilepsies and neuronal migration disorders. What remains unclear is whether there are intrinsic features of the individual dysplastic cells that give rise to heightened seizure susceptibility, or whether these dysplastic cells contribute to seizure activity by establishing abnormal circuits that alter the balance of inhibition and excitation. Mice lacking a functional p35 gene provide an ideal model in which to address these questions, because these knock-out animals not only exhibit aberrant neuronal migration but also demonstrate spontaneous seizures. Extracellular field recordings from hippocampal slices, characterizing the input-output relationship in the dentate, revealed little difference between wild-type and knock-out mice under both normal and elevated extracellular potassium conditions. However, in the presence of the GABAA antagonist bicuculline, p35 knock-out slices, but not wild-type slices, exhibited prolonged depolarizations in response to stimulation of the perforant path. There were no significant differences in the intrinsic properties of dentate granule cells (i.e., input resistance, time constant, action potential generation) from wild-type versus knock-out mice. However, antidromic activation (mossy fiber stimulation) evoked an excitatory synaptic response in over 65% of granule cells from p35 knock-out slices that was never observed in wild-type slices. Ultrastructural analyses identified morphological substrates for this aberrant excitation: recurrent axon collaterals, abnormal basal dendrites, and mossy fiber terminals forming synapses onto the spines of neighboring granule cells. These studies suggest that granule cells in p35 knock-out mice contribute to seizure activity by forming an abnormal excitatory feedback circuit.

Models of Pediatric Epilepsies: Strategies and Opportunities

Pediatric epilepsies are among the most devastating of neurologic disorders. The developing brain is particularly susceptible to seizures, and seizure activity early in brain development can cause profound neurologic impairment, enhance subsequent seizure propensity during maturation and in adulthood, and lead to abnormalities in cognitive function (1). In infancy and childhood, two broad categories of epilepsy are of particular concern because of their intractability to treatment and association with cognitive decline: (a) The so-called “catastrophic” childhood epilepsies (including infantile spasms, Lennox–Gastaut syndrome, and the progressive myoclonic epilepsies) are characterized by numerous etiologies, by age-specific developmental windows of seizure onset, by refractoriness to medical treatment, and by progressive cognitive deterioration (epileptic encephalopathy). Seizures associated with catastrophic epilepsy syndromes tend to have bilateral/generalized manifestations: (b) Refractory partial epilepsies are often associated with dysplastic brain lesions (i.e., tuberous sclerosis complex, TSC), or severe perinatally induced injuries (i.e., perinatal stroke or hypoxia); seizures of this type may be also seen with mesial temporal lobe epilepsy, but this latter condition is more prevalent in late childhood and adolescence. Often an overlap exists between these two broad categories of medically intractable epilepsies; for example, TSC is commonly associated with generalized infantile spasms as well as with multifocal partial seizures. This overlap suggests that common features may exist in the pathogenesis of different epilepsy types during early brain development. Regardless of the epilepsy type, etiology, or syndrome, the mechanisms of epilepsies in infancy and early childhood likely differ significantly from those of epilepsies in older children and adults. These mechanistic differences have important implications for therapeutic strategy and therapeutic efficacy (2,3). The lack of appropriate animal models is a major impediment to a more complete understanding of pediatric epilepsy and to more effective treatments. Animal models serve numerous functions. They provide opportunities to investigate and elucidate basic mechanisms, to test and/or develop new antiepileptic medications (AEDs) and other therapeutic modalities, to devise new diagnostic approaches, and to study the neurologic consequences of seizures at various stages of brain development (4–7). Given the many (and often significant) differences between human and animal (i.e., rodent) brain structure and developmental profiles, no animal model is likely to reproduce faithfully every aspect of a human epilepsy syndrome. However, the insightful use of such models can play a pivotal role in generating hypotheses about the mechanisms, pathogenesis, and consequences of seizures in the developing brain, and for testing potential therapies. This report summarizes discussions held during a workshop on Models of Pediatric Epilepsies, held in Bethesda, MD, on May 13–14, 2004. The Workshop was sponsored by NINDS/NIH and supported by grants from the American Epilepsy Society and the International League Against Epilepsy. Whereas previous NIH workshops have examined general priorities for epilepsy research (8,9), the current Workshop focused explicitly on epilepsies in the developing brain. Invited participants included clinical pediatric epileptologists, basic epilepsy researchers, and developmental neurobiologists (Appendix A).

Hyperexcitability of CA3 pyramidal cells in mice lacking the potassium channel subunit Kv1.1.

Summary:  Purpose: To investigate further the membrane properties and postsynaptic potentials of the CA3 pyramidal cells in mice that display spontaneous seizures because of a targeted deletion of the Kcna1 potassium channel gene (encoding the Kv1.1 protein subunit).

Morphology of cerebral lesions in the Eker rat model of tuberous sclerosis.

Tuberous sclerosis (TSC) is an autosomal dominant disorder, caused by mutations of either the TSC1 or TSC2 gene. Characteristic brain pathologies (including cortical tubers and subependymal hamartomas/giant astrocytomas) are thought to cause epilepsy, as well as other neurological dysfunction. The Eker rat, which carries a spontaneous germline mutation of the TSC2 gene (TSC2+/−), provides a unique animal model in which to study the relationship between TSC cortical pathologies and epilepsy. In the present study, we have analyzed the seizure propensity and histopathological features of a modified Eker rat preparation, in which early postnatal irradiation was employed as a “second hit” stimulus in an attempt to exacerbate cortical malformations and increase seizure propensity. Irradiated Eker rats had a tendency toward lower seizure thresholds (latencies to flurothyl-induced seizures) than seen in non-irradiated Eker rats (significant difference) or irradiated wild-type rats (non-significant difference). The majority of irradiated Eker rats exhibited dysplastic cytomegalic neurons and giant astrocyte-like cells, similar to cytopathologies observed in TSC lesions of patients. The most prominent features in these brains were hamartoma-like lesions involving large eosinophilic cells, similar to giant tuber cells in human TSC. In some cells from these hamartomas, immunocytochemistry revealed features of both neuronal and glial phenotypes, suggesting an undifferentiated or immature cell population. Both normal-appearing and dysmorphic neurons, as well as cells in the hamartomas, exhibited immunopositivity for tuberin, the protein product of the TSC2 gene.

Structural Consequences of Kcna1 Gene Deletion and Transfer in the Mouse Hippocampus

Purpose: Mice lacking the Kv1.1 potassium channel α subunit encoded by the Kcna1 gene develop recurrent behavioral seizures early in life. We examined the neuropathological consequences of seizure activity in the Kv1.1−/− (knock‐out) mouse, and explored the effects of injecting a viral vector carrying the deleted Kcna1 gene into hippocampal neurons.

Deletion of the Kv2.1 delayed rectifier potassium channel leads to neuronal and behavioral hyperexcitability

The Kv2.1 delayed rectifier potassium channel exhibits high‐level expression in both principal and inhibitory neurons throughout the central nervous system, including prominent expression in hippocampal neurons. Studies of in vitro preparations suggest that Kv2.1 is a key yet conditional regulator of intrinsic neuronal excitability, mediated by changes in Kv2.1 expression, localization and function via activity‐dependent regulation of Kv2.1 phosphorylation. Here we identify neurological and behavioral deficits in mutant (Kv2.1−/−) mice lacking this channel. Kv2.1−/− mice have grossly normal characteristics. No impairment in vision or motor coordination was apparent, although Kv2.1−/− mice exhibit reduced body weight. The anatomic structure and expression of related Kv channels in the brains of Kv2.1−/− mice appear unchanged. Delayed rectifier potassium current is diminished in hippocampal neurons cultured from Kv2.1−/− animals. Field recordings from hippocampal slices of Kv2.1−/− mice reveal hyperexcitability in response to the convulsant bicuculline, and epileptiform activity in response to stimulation. In Kv2.1−/− mice, long‐term potentiation at the Schaffer collateral – CA1 synapse is decreased. Kv2.1−/− mice are strikingly hyperactive, and exhibit defects in spatial learning, failing to improve performance in a Morris Water Maze task. Kv2.1−/− mice are hypersensitive to the effects of the convulsants flurothyl and pilocarpine, consistent with a role for Kv2.1 as a conditional suppressor of neuronal activity. Although not prone to spontaneous seizures, Kv2.1−/− mice exhibit accelerated seizure progression. Together, these findings suggest homeostatic suppression of elevated neuronal activity by Kv2.1 plays a central role in regulating neuronal network function.

Postsynaptic contributions to hippocampal network hyperexcitability induced by chronic activity blockade in vivo

Neuronal activity is thought to play an important role in refining patterns of synaptic connectivity during development and in the molecular maturation of synapses. In experiments reported here, a 2‐week infusion of tetrodotoxin (TTX) into rat hippocampus beginning on postnatal day 12 produced abnormal synchronized network discharges in in vitro slices. Discharges recorded upon TTX washout were called ‘minibursts’, owing to their small amplitude. They were routinely recorded in area CA3 and abolished by CNQX, an AMPA receptor antagonist. Because recurrent excitatory axon collaterals remodel and glutamate receptor subunit composition changes after postnatal day 12, experiments examined possible TTX‐induced alterations in recurrent excitation that could be responsible for network hyperexcitability. In biocytin‐labelled pyramidal cells, recurrent axon arbors were neither longer nor more highly branched in the TTX infusion site compared with saline‐infused controls. However, varicosity size and density were increased. Whereas most varicosities contained synaptophysin and synaptic vesicles, many were not adjacent to postsynaptic specializations, and thus failed to form anatomically identifiable synapses. An increased pattern of excitatory connectivity does not appear to explain network hyperexcitability. Quantitative immunoblots also indicated that presynaptic markers were unaltered in the TTX infusion site. However, the postsynaptic AMPA and NMDA receptor subunits, GluR1, NR1 and NR2B, were increased. In electrophysiological studies EPSPs recorded in slices from TTX‐infused hippocampus had an enhanced sensitivity to the NR2B containing NMDA receptor antagonist, ifenprodil. Thus, increases in subunit protein result in alterations in the composition of synaptic NMDA receptors. Postsynaptic changes are likely to be the major contributors to the hippocampal network hyperexcitability and should enhance both excitatory synaptic efficacy and plasticity.

BOLD-fMRI of PTZ-induced seizures in rats

PURPOSE To develop a non-invasive method for exploring seizure initiation and propagation in the brain of intact experimental animals. METHODS We have developed and applied a model-independent statistical method--Hierarchical Cluster Analysis (HCA)--for analyzing BOLD-fMRI data following administration of pentylenetetrazol (PTZ) to intact rats. HCA clusters voxels into groups that share similar time courses and magnitudes of signal change, without any assumptions about when and/or where the seizure begins. RESULTS Epileptiform spiking activity was monitored by EEG (outside the magnet) following intravenous PTZ (IV-PTZ; n=4) or intraperitoneal PTZ administration (IP-PTZ; n=5). Onset of cortical spiking first occurred at 29+/-16 s (IV-PTZ) and 147+/-29 s (IP-PTZ) following drug delivery. HCA of fMRI data following IV-PTZ (n=4) demonstrated a single dominant cluster, involving the majority of the brain and first activating at 27+/-23s. In contrast, IP-PTZ produced multiple, relatively small, clusters with heterogeneous time courses that varied markedly across animals (n=5); activation of the first cluster (involving cortex) occurred at 130+/-59 s. With both routes of PTZ administration, the timing of the fMRI signal increase correlated with onset of EEG spiking. CONCLUSIONS These experiments demonstrate that fMRI activity associated with seizure activity can be analyzed with a model-independent statistical method. HCA indicated that seizure initiation in the IV- and IP-PTZ models involves multiple regions of sensitivity that vary with route of drug administration and that show significant variability across animal subjects. Even given this heterogeneity, fMRI shows clear differences that are not apparent with typical EEG monitoring procedures, in the activation patterns between IV and IP-PTZ models. These results suggest that fMRI can be used to assess different models and patterns of seizure activation.

Are developmental dysplastic lesions epileptogenic

Cortical dysplasia of various types, reflecting abnormalities of brain development, have been closely associated with epileptic activities. Yet, there remains considerable discussion about if/how these structural lesions give rise to seizure phenomenology. Animal models have been used to investigate the cause–effect relationships between aberrant cortical structure and epilepsy. In this article, we discuss three such models: (1) the Eker rat model of tuberous sclerosis, in which a gene mutation gives rise to cortical disorganization and cytologically abnormal cellular elements; (2) the p35 knockout mouse, in which the genetic dysfunction gives rise to compromised cortical organization and lamination, but in which the cellular elements appear normal; and (3) the methylazoxymethanol‐exposed rat, in which time‐specific chemical DNA disruption leads to abnormal patterns of cell formation and migration, resulting in heterotopic neuronal clusters. Integrating data from studies of these animal models with related clinical observations, we propose that the neuropathologic features of these cortical dysplastic lesions are insufficient to determine the seizure‐initiating process. Rather, it is their interaction with a more subtly disrupted cortical “surround” that constitutes the circuitry underlying epileptiform activities as well as seizure propensity and ictogenesis.